Chemotherapy-Induced Tumor Cell Death at the Crossroads Between Immunogenicity and Immunotolerance: Focus on Acute Myeloid Leukemia

In solid tumors and hematological malignancies, including acute myeloid leukemia, some chemotherapeutic agents, such as anthracyclines, have proven to activate an immune response via dendritic cell-based cross-priming of anti-tumor T lymphocytes. This process, known as immunogenic cell death, is characterized by a variety of tumor cell modifications, i.e., cell surface translocation of calreticulin, extracellular release of adenosine triphosphate and pro-inflammatory factors, such as high mobility group box 1 proteins. However, in addition to with immunogenic cell death, chemotherapy is known to induce inflammatory modifications within the tumor microenvironment, which may also elicit immunosuppressive pathways. In particular, DCs may be driven to acquire tolerogenic features, such as the overexpression of indoleamine 2,3-dioxygensase 1, which may ultimately hamper anti-tumor T-cells via the induction of T regulatory cells. The aim of this review is to summarize the current knowledge about the mechanisms and effects by which chemotherapy results in both activation and suppression of anti-tumor immune response. Indeed, a better understanding of the whole process underlying chemotherapy-induced alterations of the immunological tumor microenvironment has important clinical implications to fully exploit the immunogenic potential of anti-leukemia agents and tune their application

Long-Term Outcome After Adoptive Immunotherapy With Natural Killer Cells: Alloreactive NK Cell Dose Still Matters

Recently, many reports were published supporting the clinical use of adoptivelytransferred natural killer (NK) cells as a therapeutic tool against cancer, including acutemyeloid leukemia (AML). Our group demonstrated promising clinical response usingadoptive immunotherapy with donor-derived alloreactive KIR-ligand-mismatched NK cellsin AML patients. Moreover, the antileukemic effect was correlated with the dose of infusedalloreactive NK cells (“functional NK cell dose”). Herein, we update the results of ourprevious study on a cohort of adult AML patients (median age at enrollment 64) infirstmorphological complete remission (CR), not eligible for allogeneic stem celltransplantation. After an extended median follow-up of 55.5 months, 8/16 evaluablepatients (50%) are still off-therapy and alive disease-free. Overall survival (OS) and disease-free survival (DFS) are related with the dose of infused alloreactive NK cells (≥2×105/kg

Sunitinib Exerts In Vitro Immunomodulatory Activity on Sarcomas via Dendritic Cells and Synergizes With PD-1 Blockade

Background: High-grade sarcomas are a heterogeneous group of aggressive tumors arising in bone and soft tissues. After relapse, treatment options are limited. The multi targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and inhibitor of PD-1 (anti-PD 1) nivolumab have shown antitumor activity in selected subtypes. In this study, we examine the role of TKIs and PD-1 based therapy in in vitro cocultures of sarcoma. Methods: The human osteosarcoma (SaOS-2) and synovial sarcoma (SYO-1) cell lines were treated with sunitinib. After cell death and proliferation assessment, expression of PD-L1 was analyzed by flow cytometry. Sunitinib-treated sarcoma cells were cocultured with dendritic cells (DCs), and the phenotype of mature DCs was determined by flow cytometry. Mature DCs were cultured with autologous T cells. PD-1 expression on T cells, their proliferation, T regulatory cell (Tregs) induction and IFN-g production, before and after nivolumab exposure, were analyzed. Results: Along with its anti-proliferative and direct pro-apoptotic effect on sarcoma cell lines, sunitinib prompted PD-L1 upregulation on sarcoma cells. Interestingly, sunitinib treated sarcoma cells drive DCs to full maturation and increase their capacity to induce sarcoma-reactive T cells to produce IFN-g. Conversely, no effect on T cell proliferation and T cell subpopulation composition was observed. Moreover, both bone and synovial sarcoma cell lines induced Tregs through DCs but sunitinib treatment completely abrogated Treg induction. Finally, sarcoma cell lines induced PD-1 upregulation on both effector T cells and Tregs when loaded into DCs, providing a rationale for using PD-1 blockade. Indeed, PD-1 blockade by nivolumab synergized with sunitinib in inducing IFN-g-producing effector T cells. Conclusions: Taken together, our in vitro data indicate that the treatment of sarcoma cells with sunitinib can exert significant changes on immune cell subsets toward immune activation, leading to DC-based cross-priming of IFN-g-producing effector T cells and reduced Treg induction. PD-1 blockade with nivolumab has a synergistic effect with sunitinib, supporting the use of TKI and anti-PD-1 approach in sarcomas, and perhaps in other cancers. DC-targeted drugs, including toll-like receptor 3 inhibitors and CD47 inhibitors, are under development and our preclinical model might help to better design their clinical application.This study was supported by the Associazione Onlus ‘il Pensatore: Matteo Amitrano’, by Bologna AIL (Associazione Italiana contro le Leucemie), Section of Bologna; FATRO, Foundation Corrado and Bruno Maria Zaini-BolognaYe

Generation of myeloma-specific T cells using dendritic cells loaded with MUC1- and hTERT- drived nonapeptides or myeloma cell apoptotic bodies.

Dendritic cells are able to induce anti-tumor immune responses by presenting tumor-specific antigens to T-lymphocytes. Various tumor-associated antigens have been studied in multiple myeloma in an effort to find a strong antigen capable of generating clinically meaningful responses in vaccinated patients. The aim of our study was to generate myeloma-specific cytotoxic T lymphocytes in vitro using dendritic cells loaded with peptide antigens or apoptotic bodies. Peripheral blood mononuclear cells from HLA-A2+ healthy donors were used for isolation and culture of dendritic cells (DCs) and T lymphocytes. DCs were loaded with hTERT- and MUC1-derived nonapeptides or apoptotic bodies from myeloma cells. Repeated stimulation of T lymphocytes led to their activation characterized by interferon-gamma production. Activated T lymphocytes were separated immunomagnetically and expanded in vitro. Specific cytotoxicity of the expanded T lymphocytes was tested against a myeloma cell line. There was evidence of cytotoxicity for all three types of antigens used for T lymphocyte priming and expansion. No statistically significant differences were observed in T lymphocyte cytotoxicity for any of the antigens. We present a method for the priming and expansion of myeloma-specific T lymphocytes using dendritic cells loaded with different types of tumor antigens. Cytotoxic T lymphocytes and/or activated dendritic cells generated by the described methods can be applied for cellular immunotherapy against multiple myeloma and other malignancies

Flow cytometry analysis of T-cell subsets in cerebrospinal fluid of narcolepsy type 1 patients with long-lasting disease

Background Type 1 narcolepsy (NT1) is a central hypersomnia linked to the destruction of hypocretin-producing neurons. A great body of genetic and epidemiological data points to likely autoimmune disease aetiology. Recent reports have characterized peripheral blood T-cell subsets in NT1, whereas data regarding the cerebrospinal fluid (CSF) immune cell composition are lacking. The current study aimed to characterize the T-cell and natural killer (NK) cell subsets in NT1 patients with long disease course. Methods Immune cell subsets from CSF and peripheral blood mononuclear cell (PBMC) samples were analysed by flow cytometry in two age-balanced and sex-balanced groups of 14 NT1 patients versus 14 healthy controls. The frequency of CSF cell groups was compared with PBMCs. Non-parametric tests were used for statistical analyses. Results The NT1 patients did not show significant differences of CSF immune cell subsets compared to controls, despite a trend towards higher CD4+ terminally differentiated effector memory T cells. T cells preferentially displayed a memory phenotype in the CSF compared to PBMCs. Furthermore, a reduced frequency of CD4+ terminally differentiated effector memory T cells and an increased frequency of NK CD56bright cells was observed in PBMCs from patients compared to controls. Finally, the ratio between CSF and peripheral CD4+ terminally differentiated effector memory T cells was two-fold increased in NT1 patients versus controls. Conclusions Significant differences in PBMCs and in CSF/PBMC ratios of immune cell profile were found in NT1 patients compared to healthy controls. These differences might have arisen from the different HLA status, or be primary or secondary to hypocretin deficiency. Further functional studies in patients close to disease onset are required to understand NT1 pathophysiology